This invention relates to a method of transforming eukaryotic cells. More particularly, this invention relates to the use of DNA constructs designed to insert a particular DNA fragment into a chosen locus of the host cell's DNA.
Through the use of recombinant DNA technology, foreign DNA sequences can be inserted into an organism's genome to alter the phenotype of the organism. A variety of different procedures have been described and utilized to produce stably transformed eukaryotic cells. All of these procedures are based on first introducing the foreign DNA into the eukaryotic cell, followed by isolation of those cells containing the foreign DNA integrated into the eukaryotic cell's DNA.
Unfortunately, all current higher eukaryotic cell transformation procedures produce transformed cells that contain the introduced foreign DNA inserted randomly and throughout the genome and in multiple copies. This random insertion of introduced DNA into the genome of host cells can be lethal if the foreign DNA happens to insert into, and thus mutate, a critically important native gene. In addition, even if the random insertion event does not impair the functioning of a host cell gene, the expression of an inserted foreign gene may be influenced by "position effects" caused by the surrounding genomic DNA. Thus a gene could be inserted into a site where the position effects are strong enough to prevent the synthesis of an effective amount of product from the introduced gene. Finally, because over-production of a gene product can have deleterious effects on the cell, genetic engineers typically wish to control the copy number of the inserted DNA, often only desiring the insertion of a single copy of the DNA sequence.
For these reasons a transformation system is desired that allows a gene to be targeted to a specific site of the host's genome. Preferably, such a system would also provide a means of preventing or subsequently removing any randomly inserted DNA sequences.
The present invention enables the targeting of a length of DNA to a specific non-lethal site in the host cell's genome, and provides for the removal of any randomly inserted DNA sequences. In addition, a proper selection of the targeted site can minimize position effects, enabling an inserted gene to synthesize an effective amount of its protein product. Therefore, this invention allows a much more precise and effective system of genetic engineering than is currently possible.
Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of the invention as presently perceived.